RCAN1 Inhibits Host Inflammatory Response To Salmonella Typhimurium Intestinal Infection

Salmonella is a gram-negative facultative intracellular bacterial pathogen, an important food-borne pathogen, that is a leading cause of enteric diease in humans and animal hosts. The acute enteritis is characterized by vomiting, anorexia, diarrhea and dehydration, even can lead to serious sepsis. Salmonella is taken up via contaiminated food and water and can infect a broad range of hosts. Salmonella comprises over 2000 serovars, of which certain host-adapted serovars can even cause a severe systemic disease which can cause high morbidity and mortality. For example,the human restricted S.enterica serovar Typhi causes Typhoid fever, which affects over 20 million people worldwide and leads to approximately 200,000 deaths per year.Salmonella Typhimurium is the main serovar of Salmonella, which is the main cause of intestinal disease in humans and animal hosts. Importantly, the pathology associated with Salmonella Typhimurium infection of mice closely resembles that of S.typhi in humans. Hence, the Salmonella Typhimurium are used to study the pathogenesis of human typhoid fever.Regulator of calcineurin 1(RCAN1) is generally regarded as an endogenous inhibitor of calcineurin. RCAN1 proteins physically interact with calcineurin subunit A and inhibit calcineurin activity, thus hampers NFAT nuclear translocation and activation of its target genes’ transcription. However, the biological functions of RCAN1 is not limited to this. Previous studies have revealed a potential role for RCAN1 during host against pathogen invasion. However, the biological implications of RCAN1 during the intestinal inflammation induced by Salmonella Typhimurium remain poorly understood. In this study, we used a Salmonella Typhimurium induced colitis model to examine the role of RCAN1 during the intestinal infection. Our results showed that the RCAN1-/-mice led to more sereve gross appearance of cecum compared to the WT counterparts early in infection, mainly contains obvious shrinkage and edema of the cecum. Subsequnently, we analyzed the baterial load inthe MLN, cecum and the pathological change of the cecum. The results showed that the baterical load in tissue of RCAN1-/-mice were significantly increased compared to WT counterparts. Extensive pathological changes and intestinal inflammation observed in the RCAN1-/-mice, which represented by increased goblet cell depletion,marked edema, infiltration of PMNs and various inflammatory cells in the submucosa and lamina propria. Subsequnently, we analyzed the cytokines and chemokines secretion of the cecum tissues. More significantly, the pro-inflammatory cytokines and chemokines were significantly increased in the cecum of RCAN1-/-mice.These results showed that RCAN1 can inhibit host inflammatory response to Salmonella Typhimurium intestinal infection.Then, we investigated the signal transduction mechanism during RCAN1 regulated the inflammatory response. It is well known that mulitiple signaling pathways have involved in the inflammatory response triggered by Salmonella Typhimurium via regulating the secretion of cytokine, of which MAPK signaling pathways also play an important role in this process. First, we analyzed the MAPK signaling pathway activation of cecum. Our results showed that phosphorylation of JNK activation was markedly increased in the cecum of RCAN1-/- mice, whereas phosphorylation of ERK and p38 did not appear significantly different between WT and RCAN1-/- mice. The results of immunohistochemical showed that RCAN1 was primarily expressed in the various inflammatory cells which infiltrated to the submucosa and lamina propria of cecum, rather than epithelial cells. Then we determined the BMDM cell as a cell model in vitro to analyze the role of RCAN1 in MAPK signal activation and the secretion of cytokine. Consistent with the results obtained in vivo, the results of in vitro experiments showed that RCAN1-/-BMDM exhibited significantly increased levels of JNK phosphorylation at indicated times compared with WT BMDM, phosphorylation of ERK and p38 did not appear significantly different between WT and RCAN1-/- BMDM. More significantly, the pro-inflammatory cytokines of IL-1β、TNF-α and chemokines of KC、CCL2 were significantly increased in the BMDM of RCAN1-/- mice. The induction of IL-1β,TNFα, KC, CCL2 is dependent on the JNK signaling pathway.Subsequently, we analyed the role of inflammatory response in controlling Salmonella Typhimurium replication in organs via survival experiment and CFU assay.The results showed that RCAN1-/- mice display greatly increased survival comparedto WT counterparts. There was significantly more bacteria detected in the liver, spleen,MLN and cecum of WT mice compared with RCAN1-/-mice at day 5 after infection.Therfore, we suggest that RCAN1 mediated downregulation of proinflammatory molecules prevented efficient clearance of Salmonella Typhimurium..Taken together, our results reveal that RCAN1 inhibits inflammatory response to Salmonella Typhimurium intestinal infection via negative regulating JNK signaling pathway, and ultimately impeding clearance of Salmonella Typhimurium...